Indole-Induced Reversion of IntrinsicMultiantibiotic Resistance in Lysobacterenzymogenes

Yong Han,a,b Yan Wang,b,c Yameng Yu,c Haotong Chen,b Yuemao Shen,a

Liangcheng Dua,b

State Key Laboratory of Microbial Technology and Key Laboratory of Chemical Biology, College of Life Sciencesand School of Pharmaceutical Sciences, Shandong University, Jinan, Chinaa; Department of Chemistry,University of Nebraska—Lincoln, Lincoln, Nebraska, USAb; College of Marine Life Sciences, Ocean University ofChina, Qingdao, Chinac

ABSTRACT Lysobacter species are a group of environmental bacteria that areemerging as a new source of antibiotics. One characteristic of Lysobacter is intrinsicresistance to multiple antibiotics, which had not been studied. To understand the re-sistance mechanism, we tested the effect of blocking two-component regulatory sys-tems (TCSs) on the antibiotic resistance of Lysobacter enzymogenes, a prolific pro-ducer of antibiotics. Upon treatment with LED209, an inhibitor of the widespreadTCS QseC/QseB, L. enzymogenes produced a large amount of an unknown metabo-lite that was barely detectable in the untreated culture. Subsequent structural eluci-dation by nuclear magnetic resonance (NMR) unexpectedly revealed that the metab-olite was indole. Indole production was also markedly induced by adrenaline, aknown modulator of QseC/QseB. Next, we identified two TCS genes, L. enzymogenesqseC (Le-qseC) and Le-qseB, in L. enzymogenes and found that mutations of Le-qseCand Le-qseB also led to a dramatic increase in indole production. We then chemicallysynthesized a fluorescent indole probe that could label the cells. While the Le-qseB(cytoplasmic response regulator) mutant was clearly labeled by the probe, the Le-qseC (membrane sensor) mutant was not labeled. It was reported previously that in-dole can enhance antibiotic resistance in bacteria. Therefore, we tested if the dra-matic increase in the level of indole production in L. enzymogenes upon blocking ofLe-qseC and Le-qseB would lead to enhanced antibiotic resistance. Surprisingly, wefound that indole caused the intrinsically multiantibiotic-resistant bacterium L. enzy-mogenes to become susceptible. Point mutations at conserved amino acids in Le-QseC also led to antibiotic susceptibility. Because indole is known as an interspeciessignal, these findings may have implications.

IMPORTANCE The environmental bacterium Lysobacter is a new source of antibioticcompounds and exhibits intrinsic antibiotic resistance. Here, we found that the inac-tivation of a two-component regulatory system (TCS) by an inhibitor or by gene de-letion led to a remarkable increase in the level of production of a metabolite in L.enzymogenes, and this metabolite was identified to be indole. We chemically synthe-sized a fluorescent indole probe and found that it could label the wild type and amutant of the TCS cytoplasmic response regulator but not a mutant of the TCSmembrane sensor. Indole treatment caused the intrinsically multidrug-resistant bac-terium L. enzymogenes to be susceptible to antibiotics. Mutations of the TCS sensoralso led to antibiotic susceptibility. Because indole is known as an interspecies signalbetween gut microbiota and mammalian hosts, the observation that indole couldrender intrinsically resistant L. enzymogenes susceptible to common antibiotics mayhave implications.

Received 2 May 2017 Accepted 12 June 2017

Accepted manuscript posted online 16June 2017

Citation Han Y, Wang Y, Yu Y, Chen H, Shen Y,Du L. 2017. Indole-induced reversion of intrinsicmultiantibiotic resistance in Lysobacterenzymogenes. Appl Environ Microbiol 83:e00995-17. https://doi.org/10.1128/AEM.00995-17.

Editor Harold L. Drake, University of Bayreuth

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Yuemao Shen,yshen@sdu.edu.cn, or Liangcheng Du,ldu3@unl.edu.

Y.H. and Y.W. contributed equally to this work.

ENVIRONMENTAL MICROBIOLOGY

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KEYWORDS indole, intrinsic antibiotic resistance, Lysobacter, natural products, two-component regulatory systems

The search for new antibiotics is a pressing and continual need, as drug-resistantpathogens constantly emerge. Natural products are a main source of antibiotics,

and Gram-positive bacteria of the Actinomycetes and filamentous fungi have been theprimary sources of bioactive natural products for decades. In recent years, the Gram-negative bacterium Lysobacter is emerging as a new source of antibiotic naturalproducts with new structural features and interesting modes of action (1, 2). Forexample, lysocins from Lysobacter sp. strain RH2180-5 are cyclic peptide antibiotics thatexhibit a novel mode of action by binding to menaquinone in the cell membranes (3,4). Lysobactin (katanosin B) from Lysobacter sp. strain ATCC 53042 is a potent antibac-terial cyclic depsipeptide that binds to the reducing end of lipid-linked cell wallprecursors and thus blocks peptidoglycan biosynthesis of the cell wall (5–7). WAP-8294A from Lysobacter sp. strain WAP-8294 and Lysobacter enzymogenes OH11 is afamily of cyclic lipodepsipeptides with potent activity against methicillin-resistantStaphylococcus aureus (8–10). Tripropeptins from Lysobacter sp. strain BMK333-48F3 areanother group of cyclic lipodepsipeptides that inhibit the lipid cycle of bacterial cellwall biosynthesis by forming a complex with Ca2� and undecaprenyl pyrophosphate(11, 12). Cephabacins from Lysobacter lactamgenus contain a cephem core attached toan oligopeptide side chain (13–15). Heat-stable antifungal factor (HSAF) and analogsfrom L. enzymogenes strains C3 and OH11 are a group of polycyclic tetramate macrolac-tams with potent antifungal activity and a distinct mode of action (16–24). Phenazines fromLysobacter antibioticus OH13 are broad-spectrum antibiotics, including the well-knownantibiotic myxin (25).

As ubiquitous environmental Gram-negative bacteria, Lysobacter species have twodistinct characteristics. One is their unusual richness in peptide metabolites, and theother is their intrinsic resistance to multiple antibiotics. For the former characteristic, weand others have started to look into molecular mechanisms for the production andregulation of bioactive natural products. Several Lysobacter genomes have been se-quenced, and most of these genomes contain multiple gene clusters (12 to 16 clusters)for natural product biosynthesis (2, 26). Unlike gene clusters of Gram-positive Strepto-myces species that are also rich in bioactive natural products, most of the Lysobactergene clusters are predicted to synthesize peptides. Lysobacter species are considered“peptide production specialists” (2). In addition, recent studies have shown that theproduction of antibiotic compounds is regulated by endogenous and/or xenogenoussmall molecules (27–31). For example, L. enzymogenes diffusible signaling factor 1(LeDSF1) to LeDSF5, a group of fatty acids, were identified in L. enzymogenes strainOH11 and found to act as DSFs to regulate the biosynthesis of HSAF (27). The signalingis mediated by a two-component regulatory system (TCS), L. enzymogenes RpfC (Le-RpfC)/Le-RpfG, which is responsible for sensing the DSF and for conveying the signal forsubsequent gene expression and HSAF production. Independent of DSF signaling,4-hydroxbenzoic acid was recently shown to serve as a diffusible factor (DF) to regulateHSAF biosynthesis through a LysR family transcription factor (LysRLe) (28–31). Interest-ingly, PilS/PilR, another TCS that is known to regulate T4P (type IV pilus) production inmany bacteria, was unexpectedly found to play a role in HSAF regulation in L.enzymogenes, and signaling was conveyed by the second messenger cyclic di-GMP(c-di-GMP) (32).

In contrast to the biosynthesis and regulation of natural products, essentiallynothing is known about the molecular mechanism(s) underlying intrinsic multidrugresistance in Lysobacter. An understanding of the mechanism(s) is important for thebetter utilization of this largely underexplored source of bioactive natural products. Inthis work, we serendipitously found that a simple molecule, indole, is able to reversethe antibiotic resistance of Lysobacter through a QseC (quorum sensing Escherichia coliregulator C)/QseB-like TCS. QseC/QseB was originally found in E. coli (33, 34). QseC is the

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membrane-bound sensor protein of the TCS with histidine kinase activity, and QseB isthe cytoplasmic response regulator that regulates the expression of downstream targetgenes. QseC/QseB homologs are present in many important human and plant patho-gens and are important for the virulence of pathogens (35–37). In enterohemorrhagicE. coli, QseC/QseB regulates multiple virulence factors and is known to respond tohuman epinephrine/norepinephrine and bacterial AI-3 (autoinducer 3), a group ofmysterious signals whose identity is yet to be discovered (35–40). QseC is considereda target for new antibiotic development, and a potent QseC inhibitor, LED209, wasidentified (35, 41, 42). Through QseC/QseB signaling, AI-3 signals regulate the expres-sion of enterocyte effacement, Shiga toxin, and flagellum and motility genes in entero-hemorrhagic E. coli (39). In this study, we describe the identification of indole as asmall-molecule signal for Le-QseC/Le-QseB in L. enzymogenes OH11 and present evi-dence for the involvement of this signaling system in intrinsic multiantibiotic resistance.

RESULTSTreatment of L. enzymogenes OH11 with the QseC inhibitor LED209 induces a

dramatic increase in indole production. In bacteria, the TCS is the basic system forsensing and responding to environmental stimuli and signaling factors (43). Sinceantibiotic treatment could be regarded as a type of environmental stress to bacteria, wewere curious about the potential consequences of blocking TCSs in L. enzymogenes.Because of the widespread occurrence of QseC/QseB and its importance in pathogenicbacteria, we chose to test LED209, a small synthetic compound that exhibited potentinhibitory activity against the binding of signals to QseC (35). After LED209 treatment,we examined the metabolite profile of L. enzymogenes OH11. The level of a newmetabolite with a 7.8-min retention time upon high-performance liquid chromatogra-phy (HPLC), which was barely detectable in untreated OH11 cells, was dramaticallyincreased in OH11 cells treated with LED209 (10 pM) (Fig. 1). We purified the compoundfrom a scale-up culture and elucidated its chemical structure using nuclear magneticresonance (NMR) (see Fig. S1 in the supplemental material). 1H-NMR (400 MHz, CDCl3)gave the following signals: � 8.28 (s, 1H), 7.67 (d, J � 7.9 Hz, 1H), 7.42 (d, J � 8.1 Hz, 1H),7.22 (m, 2H), 7.14 (t, J � 7.5 Hz, 1H), and 6.58 (s, 1H) ppm. 13C-NMR (100 MHz, CDCl3)gave the following signals: � 135.81, 127.86, 124.13, 121.94, 120.71, 119.76, 111.02, and102.57 ppm. The NMR data indicate that this compound is indole, which was confirmedby comparison with the spectra and HPLC data for standard indole.

The dramatic induction of indole by LED209 is intriguing, because indole and someof its derivatives are signal molecules involved in biofilm formation, virulence, motility,and antibiotic tolerance (44). The concentration of indole can reach �100 �M in thehuman gastrointestinal (GI) tract and 250 to 1,100 �M in human feces (45, 46). Culturedcommensal E. coli cells can produce as much as 600 �M indole (47). For L. enzymogenesOH11, we estimated that upon LED209 treatment, the concentration of indole in-creased over 1,300-fold (from approximately 0.3 �M to about 410 �M). We were ableto obtain �30 mg indole from 1 liter of culture (corresponding to approximately 256�M), confirming the drastic induction of indole production by the QseC inhibitorLED209. Finally, one hallmark of the QseC/QseB-like TCSs is that they respond toadrenergic signals such as epinephrine (adrenaline). Indeed, treatment with epineph-rine also led to a remarkable induction of indole production in L. enzymogenes OH11(Fig. 1).

Deletion of Le0754-Le0752 in L. enzymogenes OH11 also leads to a largeincrease in indole production. The remarkable induction of indole by the QseCinhibitor LED209 suggests that indole signaling may involve a QseC/QseB-like TSC in L.enzymogenes. Among the genes coding for putative TCSs in L. enzymogenes (32), wefound that Le0754-Le0752 showed the highest similarity to genes for QseC/QseB of E.coli (strain K-12 and enterohemorrhagic strain O157:H7). Le0754 shares 31.1% identityand 43.3% similarity to QseC of E. coli, and Le0752 has 47.5% identity and 62.0%similarity to QseB of E. coli (see Fig. S2 in the supplemental material). We thus use thenames Le-qseC and Le-qseB for Le0754 and Le0752, respectively, in this study. To test if

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FIG 1 Indole production in Lysobacter enzymogenes OH11. (A) Wild-type strain OH11; (B) strain OH11 treated with LED209 (10 pM); (C) strain OH11treated with epinephrine (5 mM); (D) Le-qseC deletion mutant; (E) Le-qseB deletion mutant.

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this TSC plays a role in indole signaling, we examined the expression of the Le-qseCgene and the Le-qseB gene upon exogenous indole treatment or LED209 treatment.The results showed that indole slightly upregulated Le-qseB and Le-qseC, while LED209clearly downregulated these two genes (Fig. S3). These results indicate that theinduction and subsequent accumulation of indole by LED209 do not appear to have asignificant effect on the gene expression of Le-qseB and Le-qseC.

To test whether indole signaling is mediated by Le-QseC/Le-QseB, we deleted theLe-qseC gene and the Le-qseB gene in OH11. Similarly to LED209 treatment, the deletionof this TCS also led to a remarkable increase in the level of indole production (estimatedat 267 �M for the Le-qseC mutant and 323 �M for the Le-qseB mutant) (Fig. 1). Theseresults clearly demonstrate that the loss of function of the Le-QseC/Le-QseB TCS leadsto indole induction in OH11.

Fluorescently labeled indole binds to wild-type L. enzymogenes and the Le-QseB mutant but not to the Le-QseC mutant. To obtain evidence for the involvementof Le-QseC/Le-QseB in indole signaling, we chemically synthesized a fluorescent indoleprobe (compound 1) in which indole was linked to the fluorophore Azide-fluor 488(from Sigma-Aldrich; compound 4) using click chemistry (Fig. 2). When this probe wasincubated with L. enzymogenes OH11 cells, wild-type cells and Le-qseB deletion mutantcells were clearly labeled with green fluorescence, while Le-qseC deletion mutant cellswere not labeled (Fig. 3). As controls, L. enzymogenes cells gave no fluorescence in theabsence of the probe or when the cells were incubated with the fluorophore alone(compound 4). These results suggest that indole binds to Le-QseC, the membranesensor of the TCS, but not to Le-QseB, the cytoplasmic response regulator of the TCS.

To obtain more evidence, we generated four other Le-qseC mutants, each of whichcontained a single amino acid change at four conserved residues within the N-terminalligand-binding domain (but not the C-terminal kinase domain [see Fig. S2 in thesupplemental material]). While the wild type clearly showed fluorescence, the Le-qseCD85V, Le-qseC F126A, and Le-qseC F154A single point mutants gave barely detectablefluorescence signals when incubated with the indole probe (Fig. 4). On the other hand,the Le-qseC W164E mutant exhibited fluorescence similar to that of the wild type. Dueto the lack of structural information on Le-QseC, it is difficult to predict the potentialcontributions of these conserved amino acid residues to the function of Le-QseC.Nonetheless, these data support that the binding of indole to L. enzymogenes cells isLe-QseC dependent.

FIG 2 Chemical synthesis of a fluorescent indole probe (compound 1). Indole (compound 2) was linked to the fluorophore Azide-fluor 488 (compound 4) byusing click chemistry via an alkyne intermediate (compound 3).

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Indole reverses antibiotic resistance in L. enzymogenes OH11. To evaluatepotential physiological consequences of Le-QseC/Le-QseB-mediated indole signaling,we examined antibiotic resistance in L. enzymogenes OH11, because this is one of thedistinct characteristics of Lysobacter species. First, we checked whether exogenousindole would have a toxic effect on OH11 growth. In the absence of an antibiotic, indole(0.5 mM) exhibited no obvious effect on OH11 growth in both solid culture and liquidculture (Fig. 5). In contrast, indole almost completely stopped the growth of OH11 inthe presence of several common antibiotics, although OH11 is intrinsically resistant to

FIG 3 Fluorescence imaging of Lysobacter enzymogenes OH11, the Le-qseB deletion mutant, and theLe-qseC deletion mutant. (A) Strain OH11; (B) strain OH11 treated with compound 4 (the fluorophorealone); (C) strain OH11 treated with compound 1 (the fluorophore with the indole probe); (D) Le-qseBdeletion mutant treated with compound 1; (E) Le-qseC deletion mutant treated with compound 1. Eachof the picture panels shows a picture taken at a 488-nm excitation wavelength and a 509-nm emissionwavelength (a), a picture taken under transmitted light (b), and a merged image of panels a and b (c).

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these antibiotics (Fig. 5). These results show that indole treatment causes a multiantibiotic-resistant Lysobacter strain to become susceptible.

To further look into the reversed antibiotic resistance, we examined the TCSLe-qseC–Le-qseB mutants, including gene deletion mutants and single point mutants.While the wild type grew normally in medium containing ampicillin (100 �g/ml),kanamycin (50 �g/ml), or gentamicin (5 �g/ml), the Le-qseC deletion mutant wasunable to grow under the same conditions, regardless of the presence or absence ofindole (Fig. 6). This confirms the involvement of the Le-QseC sensor of the TCS inantibiotic susceptibility. In contrast, the Le-qseB deletion mutant was able to grow,albeit more slowly than the wild type, in medium containing ampicillin, kanamycin, orgentamicin, in the absence of indole (Fig. 6). However, in the presence of indole, theLe-qseB deletion mutant became totally susceptible to the antibiotics. This is consistentwith data from the fluorescent indole binding experiments, which showed that theLe-QseB response regulator of the TCS does not directly interact with indole. As long asthe Le-QseC sensor is functional, indole signaling could be transmitted to the cells,perhaps via an alternative response regulator of the 54 putative TCSs found in OH11(32). This could explain why the Le-qseB deletion mutant grew more slowly than thewild type in the absence of indole, probably due to a less optimal cytoplasmic responseregulator for the membrane sensor Le-QseC.

To obtain more evidence, we also tested the Le-QseC single-amino-acid mutants(Fig. 6). As expected, no growth was observed for the Le-qseC D85V mutant, the Le-qseCF126A mutant, or the Le-qseC F154A mutant in medium containing ampicillin, kana-mycin, or gentamicin, with or without indole. In contrast, the Le-qseC W164E mutantgrew nearly as well as the wild type in medium containing the antibiotics but withoutindole. However, when indole was added to the medium, the Le-qseC W164E mutantcould not grow in medium containing any of the antibiotics (Fig. 6). These results arein agreement with those from fluorescent indole binding experiments with the Le-qseCsingle-amino-acid mutants.

Indole stimulates HSAF production. Because L. enzymogenes is known to produceseveral antibiotic metabolites, we examined indole’s effect on the biosynthesis of HSAF,

FIG 4 Fluorescence imaging of Lysobacter enzymogenes OH11 and Le-qseC single-amino-acid mutants. OH11 andthe mutants were treated with compound 1 (indole fluorophore probe), and images of the cells were taken at a488-nm excitation wavelength and a 509-nm emission wavelength. (A) Wild-type strain OH11; (B) Le-qseC D85Vmutant; (C) Le-qseC F126A mutant; (D) Le-qseC F154A mutant; (E) Le-qseC W164E mutant.

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the major antifungal metabolite in this strain. The exogenous addition of indolemarkedly increased the production of HSAF and its analogs in L. enzymogenes (see Fig.S6 in the supplemental material). These results suggest that HSAF biosynthesis isaffected by indole signaling.

DISCUSSION

Indole signaling in bacteria has been investigated by several groups. Early studiesreported that SdiA, a quorum sensing regulator of E. coli, mediated indole signaling, inaddition to sensing acyl homoserine lactones (44, 48, 49). Indole was shown to inducemultidrug exporter gene expression, and its signaling was transmitted via BaeSR/CpxAR, another TCS in E. coli (50). A more recent study found that while indole had

FIG 5 Effect of indole on antibiotic resistance of L. enzymogenes OH11. (A and B) Indole exhibits little effect on thegrowth of L. enzymogenes OH11 in solid (A) and liquid (B) media. OD600, optical density at 600 nm. (C) In contrast,indole nearly completely stopped the growth of L. enzymogenes OH11 in the presence of several antibiotics,although Lysobacter species are intrinsically resistant to these antibiotics.

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effects on gene expression and biofilm formation, these effects were not dependent onSdiA in E. coli and Salmonella enterica serovar Typhimurium (51). Furthermore, indolewas shown to be freely diffusible across bacterial membranes (52). Thus, it was notentirely clear whether indole signaling would require a sensor/receptor protein and, ifso, whether the protein would be a TCS-type sensor.

In this study, we found that LED209, an inhibitor of the TCS sensor QseC, andepinephrine, a known modulator of QseC, dramatically induced indole production in L.enzymogenes. We further discovered that the deletion of the Le-qseC gene or theLe-qseB gene in L. enzymogenes also remarkably stimulated indole production. Using achemically synthesized fluorescent indole probe, we obtained evidence to support thatindole could bind to the sensor protein Le-QseC in L. enzymogenes. The QseC/QseB

FIG 6 Effect of mutations in Le-qseC–Le-qseB on the antibiotic resistance of L. enzymogenes OH11 in the absence or presence of indole.(A) Le-qseC deletion mutant; (B) Le-qseB deletion mutant; (C) Le-qseC D85V mutant; (D) Le-qseC F126A mutant; (E) Le-qseC F154A mutant;(F) Le-qseC W164E mutant.

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signaling system is very important for the virulence and motility of enterohemorrhagicE. coli, which uses epinephrine/norepinephrine and AI-3 as signal molecules (35–40).However, the nature of this so-called AI-3 remains unclear, other than that AI-3 wassuggested to be a group of bacterial aromatic compounds. The QseC inhibitor LED209reduces the in vivo virulence of enterohemorrhagic E. coli and Salmonella entericaserovar Typhimurium. Targeted blocking of QseC activity has been proposed as astrategy against Gram-negative pathogens (42).

In contrast to pathogenic E. coli and Salmonella enterica, L. enzymogenes is anonpathogenic environmental bacterium. Its QseC/QseB-like system is not expected tobe associated with virulence, and our results show that this TSC is involved in antibioticresistance (Fig. 7). In E. coli, indole was shown to serve as a signal to turn on drug effluxpumps and oxidative stress-protective mechanisms, which conferred population-basedantibiotic resistance (53). Indole signaling also induced E. coli persistence throughactivating stress responses in the bacterial community (54). As an interspecies signal,indole caused Salmonella to become less susceptible to antibiotics (55). In the presenceof antibiotics, E. coli cells produced a higher level of indole that enhanced cell survivalduring antibiotic stress (56). It appears that indole generally enhances antibioticresistance in these pathogenic bacteria. Therefore, in light of the dramatic increase inindole production in L. enzymogenes upon blocking of Le-QseC or mutation of Le-qseC–Le-qseB, our first instinct was to expect enhanced antibiotic resistance. However, to oursurprise, we found that indole caused the intrinsically multidrug resistant bacterium L.enzymogenes to become susceptible to several common antibiotics. Similarly, theLe-qseC–Le-qseB mutants also became antibiotic susceptible. In the absence of anantibiotic, indole itself did not show toxicity to the growth of L. enzymogenes.

Indole has been shown to induce the production of efflux pumps in both E. coli andSalmonella (53, 57). The change in efflux pump activity could be a main reason for theobserved change in antibiotic resistance in these bacteria. However, indole’s effect is toincrease antibiotic resistance in the above-described pathogenic bacteria. For theenvironmental bacterium L. enzymogenes, we found the opposite effect, that indolecaused this intrinsically resistant bacterium become susceptible (Fig. 7). This maysuggest that the direction of antibiotic transportation by the indole-induced effluxpumps in L. enzymogenes is probably the opposite of that in the pathogenic bacteriadescribed above. The increase in the level of indole may lead to enhanced antibiotictransport from outside to inside the cells so that an inhibitory concentration of the

FIG 7 Indole-mediated reversal of multiantibiotic resistance in L. enzymogenes OH11. The two-component regulatory system Le-QseC/Le-QseB is involved in this process.

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antibiotic could be reached. Whether this is a feature specific only to the environmentalGram-negative bacterium Lysobacter or a property present in broader microorganismsis worth further investigation, particularly for members of the gut microbiota, becausethey are known to produce a large amount of indole and to be involved in diseases inhumans (45–47). In addition, QseC homologs are present in many important humanand plant pathogens, and the QseC/QseB signaling pathway is important for thevirulence of these pathogens. Thus, the finding that indole could cause an antibiotic-resistant bacterium to be antibiotic susceptible has important implications in terms ofunderstanding the pathology as well as developing potentially novel approaches towardfighting against multidrug-resistant superbugs, particularly Gram-negative pathogens,against which there are very few weapons available.

Finally, Le-QseC/Le-QseB is the third TCS among the 54 putative TCSs whose signalmolecules have been identified (27, 32). As Lysobacter species produce a large numberof bioactive natural products and their regulatory mechanism remains largely unex-plored (1, 2), it will be interesting to test if Le-QseC/Le-QseB-mediated indole signalingcould also play a role in regulating antibiotic production by Lysobacter.

MATERIALS AND METHODSBacterial strains, plasmids, and general methods. Escherichia coli strains DH5� and S17-1 were

used as the hosts for general DNA manipulation and for conjugation, respectively. Lysobacter enzymo-genes OH11 (CGMCC 1978) and the derived mutants were grown in 40%-strength tryptic soy broth (TSB).Plasmid miniprep and DNA fragment extraction were performed according to the instructions of the kitsfrom Qiagen. All other manipulations were carried out according to methods described previously (21).PCR primers were synthesized by Eurofins MWG Operon (distributed by Fisher Scientific). Restrictionenzymes and other molecular biology reagents were purchased from NEB. LED209 was purchased fromSanta Cruz Biotechnology, Azide-fluor 488 was obtained from Sigma-Aldrich, and all other generalchemicals were purchased from Sigma-Aldrich or Fisher Scientific.

Isolation and structural determination of a new signal molecule in L. enzymogenes OH11. L.enzymogenes OH11 was grown in 40% TSB (50 ml, containing 5 pM LED209) at 28°C at 200 rpm for 3 days.The culture was extracted with the same volume of ethyl acetate. The organic phase was concentratedunder a vacuum, and the residues were dissolved with 0.5 ml methanol (MeOH) to afford the crudeextract. An aliquot (20 �l) of the crude extract was injected into an HPLC instrument to analyze themetabolite prolife of the culture containing LED209, in comparison with that of the control culture. TheHPLC instrument (1120 system; Agilent, Santa Clara, CA) was equipped with a reverse-phase (RP) C18

column (4.6 by 150 mm, 3.5 �m), using the solvent system described in Table S1 in the supplementalmaterial. This led to the identification of a new peak that was dramatically increased upon LED209treatment. To identify this new compound, the culture was scaled up to 1 liter (40% TSB, 5 pM LED209)and grown at 28°C at 200 rpm for 3 days. The culture was filtered, and the filtrate was extracted with 1liter of ethyl acetate. The organic solution was collected and concentrated by using a Rotavaporinstrument. The crude extract was dissolved in methanol (2 ml) and loaded onto a column containing 10g of silica gel 60 (Merck, Darmstadt, Germany). The column was eluted with CHCl3 and MeOH (from 100%CHCl3 to 100% MeOH) to afford 5 fractions, fraction 1 (100% CHCl3), fraction 2 (CHCl3-MeOH at a 100:1dilution), fraction 3 (95% CHCl3–5% MeOH), fraction 4 (90% CHCl3–10% MeOH), and fraction 5 (100%MeOH). Thin-layer chromatography (TLC) (precoated silica gel 60 F254 plates; Merck, Darmstadt, Ger-many) was used to monitor the new compound in the fractions. The fraction (fraction 3) containing thecompound was concentrated, and the residues were redissolved in methanol (2 ml). The solution wasapplied to a preparative HPLC column (RP-C18 [10.0 by 250 mm, 5 �m]), which was developed with 50%acetonitrile. The peak containing this new metabolite was collected to obtain �30 mg of pure com-pound. To determine the chemical structure, the compound was subjected to spectral analysis by NMR(Bruker Advance 400 spectrometer, 400/100 MHz; Bruker, Fällanden, Switzerland) and mass spectrometry(MS) (liquid chromatography quadrupole [LCQ] mass spectrometer; Thermo, West Palm Beach, FL, USA).

Quantitative PCR (qPCR). The wild type and mutants of L. enzymogenes were grown on 100 ml1/10-strength TSB medium for 24 h. An aliquot of 3 ml of cells was transferred to sterile centrifuge tubesand centrifuged for 3 min at 15,300 � g. The TRIzol solution was used to isolate the total RNA from thecells according to the manufacturer’s instructions. A PrimerScript RT reagent kit with a genomic DNA(gDNA) Eraser kit (TaKaRa Biocompany) was used to remove DNA and for reverse transcription of themRNA. An iQ SYBR green supermix kit (Bio-Rad) was used to quantitatively determine the expressionlevels of target genes, with 16S RNA as the reference (27).

Chemical synthesis of the fluorescent indole probe (compound 1). Zinc dust (1.3 g; 20 mmol) wasadded to a solution containing indole 2 (585 mg; 5 mmol) and propargyl bromide (2.4 ml; 20 mmol) in6 ml of tetrahydrofuran (THF) at ambient temperature. After stirring for 12 h, the reaction mixture wasdiluted with ethyl acetate (EtOAc), filtered through a pad of Celite, and concentrated in vacuo. Quick flashchromatography (10:1 hexane-EtOAc) yielded 542 mg of the target compound 3-(prop-2-yn-1-yl)-1H-indole (compound 3) (70%), and the compound was characterized as follows: Rf � 0.87 (3:1 hexane-EtOAc); 1H NMR (400 MHz, CDCl3), � 8.03 (br s, 1H), 7.68 (d, 2H, J � 7.8 Hz), 7.39 (d, 1H, J � 8.1 Hz), 7.25(t, 1H, J � 7.6 Hz), 7.20 (s, 1H), 7.18 (t, 1H, J � 7.6 Hz), 3.76 to 3.72 (m, 2H), and 2.19 to 2.16 (m, 1H).

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A stirring solution of THF-water (1:2; 0.3 ml) at 25°C was charged with Azide-fluor 488 (compound 4)(0.574 mg; 0.001 mmol), compound 3 (0.155 mg; 0.001 mmol), copper sulfate (0.1 mg; 0.005 mmol),triethylamine (TEA) (0.05 mg; 0.005 mmol), and sodium ascorbate (0.2 mg; 0.001 mmol). The resultingmixture was stirred at room temperature for 24 h. Reaction progress was monitored by electrosprayionization (ESI)-mass spectrometry. Sephadex LH-20 chromatography (75% methanol) yielded 0.526 mgof the target compound (72%). The mass was calculated to be 729.79 for C40H40N7O7 and found m/z730.67 [M � H]� by ESI-MS. More spectral characterization of the synthetic compounds is included in Fig.S4 and S5 in the supplemental material.

Detection of fluorescence in various strains of L. enzymogenes using the synthetic indoleprobe. To test the binding of the fluorescent indole probe, various strains of L. enzymogenes, includingwild-type strain OH11, the Le-qseC and Le-qseB deletion mutants, and the Le-qseC single-amino-acid pointmutants, were grown in 3 ml LB at 30°C at 250 rpm overnight. The cells were harvested by centrifugationand resuspended in a 1-ml solution of the probe (50 �M) in phosphate-buffered saline (PBS) (pH 7.4) at4°C. The cells were then incubated at 30°C for 2 h, pelleted, and washed 2 times with PBS (pH 7.4) at 4°C.Finally, the cells were fixed by using 1/10 formaldehyde in PBS for 1 h at 25°C. The fluorescence of thecells was detected and photographed on an Olympus IX 81 inverted confocal microscope (Olympus,Tokyo, Japan). A 488-nm argon laser was utilized for continuous spectral detection. When indole bindsto Le-QseC on the cell membranes of L. enzymogenes, the cells will be labeled with green fluorescencedue to the fluorescent reporter of the probe.

Generation of in-frame Le-qseB and Le-qseC gene deletion mutants. To construct vectors for thein-frame deletion of Le-qseB and Le-qseC in L. enzymogenes OH11, two DNA fragments were amplifiedfrom the upstream and downstream regions of each of these two genes by using the primer pairs shownin Table 1. Genomic DNA from wild-type L. enzymogenes OH11 was used as the PCR template. Each ofthe upstream fragments was digested with XhoI/PstI, and each of the downstream fragments wasdigested with PstI/BamHI. The upstream and downstream fragments of Le-qseB were cloned into theconjugation vector pJQ200SK to produce pJQ200SK-qseBupdown, and those of Le-qseC were cloned intopJQ200SK to generate pJQ200-qseCupdown. The resulting vectors were transferred into L. enzymogenesaccording to procedures described previously (17), and gentamicin-resistant colonies were selected forPCR verification by using primers listed in Table 1. The confirmed colonies resulting from single-crossoverhomologous recombination were subjected to a double crossover, which led to the loss of the vectorthrough a second homologous recombination. The Le-qseB double-crossover mutants were screened byusing primers qseB-up-forw and qseB-down-revs, and the qseC double-crossover mutants were screenedby using primers qseC-up-forw and qseC-down-revs (Table 1). The qseB double-crossover mutantsgenerated the expected 620-bp fragment, and the qseC double-crossover mutants generated theexpected 833-bp fragment. Genomic DNA of wild-type L. enzymogenes and water were used as controls.

Generation of Le-qseC single-amino-acid mutants. To construct vectors for Le-qseC single-amino-acid mutants in L. enzymogenes OH11, two DNA fragments that overlap and contain a single codonchange at each of the four conserved residues in Le-qseC, as shown in Fig. S2 in the supplementalmaterial, were amplified by PCR using primer pairs (qseC-UP and qseC-DW) listed in Table 1. Genomic

TABLE 1 Primers used in this study

Primer Sequence

qseB-deletion-up-forw TGT CTC GAG CAC AGC GCG ATC AqseB-deletion-up-revs GTC CTG CAG TGT GTA TAC GGC AAqseB-deletion-down-forw GTA CTG CAG GAA CAG CTG ATC GqseB-deletion-down-revs TAA GGA TCC ACC CAG GTG AGC TTqseC-deletion-up-forw ACT CTC GAG TCA AAC CCT TCC AqseC-deletion-up-revs TAA CTG GGT TCG CCG AGC TTqseC-deletion-down-forw TAT CTG CAG TGC GAA CGC ATC TTqseC-deletion-down-revs TAA GGA TCC GAA GTC GAA GTC GTGqseB-verif-forw TGT CTG GCA CAG CGC GAT CAqseB-verif-revs TAA GGA CCA CCC AGG TGA GCT TqseC-verif-forw ATC TGA GTC AAC CCT TCC AqseC-verif-revs TAA GGA TCC GAA GTC GAA GTC GTGqseB-up-forw TGT CTC GAG CAC AGC GCG ATC AqseB-down-revs TAA GGA TCC ACC CAG GTG AGC TTqseC-up-forw ACT CTC GAG TCA AAC CCT TCC AqseC-down-revs TAA GGA TCC GAA GTC GAA GTC GTGqseC-UP AAT GGT ACC GGG CGG CCG CCGqseC-DW AAT GGA TCC CCT ACA GGG GGA AAC CGG AGA85D-RE1 GAT CTC GCG CAG GGA GTG CAC GAG CAT G85D-FW2 GAG CGG ACC GGC ATG CTC GTG CAC TCC CTG CC126F-RE1 GCC GTG CGC CCA CAC CTG AGC GCT CAT CTT CTG126F-FW2 GCC GAC CAG AAG ATG AGC GCT CAG GTG TGG GCG154F-RE1 GTC GAT CAC GCG GCG GGC GCC GCC GTC CTT GAA154F-FW2 CCC GAA TTC AAG GAC GGC GCC GCC CGC CGC164F-RE1 GGT GAG GGT GTA GAC CTG GAG GCG CTC GCC GTC164F-FW2 GTG ATC GAC GGC GAG CGC CTC CAG GTC TAC

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DNA from OH11 was used as the template, and the two fragments were pieced together through asecond PCR. Each of the resultant fragments was digested with KpnI/BamHI and then cloned into theconjugation vector pET18Gm to produce pET18Gm-qseCupdown. The constructs were confirmed bysequencing and then transferred into L. enzymogenes OH11 according to procedures described previ-ously (17), and gentamicin-resistant colonies were selected for PCR verification of single-crossoverhomologous recombination using primers listed in Table 1. The confirmed colonies resulting fromsingle-crossover homologous recombination were subjected to a double crossover, which led to the lossof the vector through a second homologous recombination. The double-crossover mutants werescreened by using test primers (qsec-UP and qsec-DW). The PCR products were digested with the selectedrestriction enzymes (ApaLI for the Le-QseC D85V mutant, AfeI for the Le-QseC F126A mutant, EheI for theLe-QseC F154A mutant, and BpmI for the Le-QseC W164E mutant) to verify the mutants.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.00995-17.

SUPPLEMENTAL FILE 1, PDF file, 0.5 MB.

ACKNOWLEDGMENTSThis work was supported in part by the NSFC (31329005), the NIH (R01AI097260), the

Nebraska Research Initiative, a UNL Research Council Faculty Seed Grant, and theTaishan Scholars Program of Shandong Province.

We thank Joe Zhou and Terri Fangman for technical assistance.L.D., H.Y., Y.W., and Y.S. conceived and designed experiments. H.Y., Y.W., Y.Y., and

H.C. carried out experiments. L.D., Y.H., and Y.W. analyzed data. L.D. wrote the manu-script. Y.H., Y.W., L.D., and Y.S. reviewed and revised the manuscript.

We declare that we have no conflicts of interest with the contents of this article.

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